Dorsal root ganglion (DRG) toxicity has been consistently reported as a potential safety concern after delivery of adeno-associated viruses (AAVs) containing gene-replacement vectors but has yet to be reported for RNAi-based vectors. Here, we report DRG toxicity after AAV intra-CSF delivery of an RNAi expression construct—artificial microRNA targeting superoxide dismutase 1 (SOD1)—in non-human primates (NHPs) and provide evidence that this can be recapitulated within mice. Histopathology evaluation showed that NHPs and mice develop DRG toxicity after AAV delivery, including DRG neuron degeneration and necrosis and nerve-fiber degeneration that were associated with increases in cerebrospinal fluid (CSF) and serum phosphorylated neurofilament heavy chain (pNF-H). RNA-sequencing analysis of DRGs showed that dysregulated pathways were preserved between NHPs and mice, including increases in innate/adaptive immune responses and decreases in mitochondrial- and neuronal-related genes, following AAV treatment. Finally, endogenous miR-21-5p was upregulated in DRGs of AAV-treated NHPs and mice. Increases in miR-21-5p were also identified within the CSF of NHPs, which significantly correlated with pNF-H, implicating miR-21-5p as a potential biomarker of DRG toxicity in conjunction with other molecular analytes. This work highlights the importance of assessing safety concerns related to DRG toxicity when developing RNAi-based AAV vectors for therapeutic purposes.

中文翻译：

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AAV 脑脊液内将 RNAi 表达构建体递送到非人灵长类动物和小鼠体内后的背根神经节毒性


背根神经节 （DRG） 毒性一直被报道为递送含有基因替代载体的腺相关病毒 （AAV） 后的潜在安全问题，但尚未报道基于 RNAi 的载体。在这里，我们报告了非人灵长类动物 （NHP） 中 RNAi 表达构建体——靶向超氧化物歧化酶 1 （SOD1） 的人工 microRNA）在 AAV 脑脊液内递送后的 DRG 毒性，并提供证据证明这可以在小鼠中概括。组织病理学评估显示，NHPs 和小鼠在 AAV 递送后发生 DRG 毒性，包括 DRG 神经元变性和坏死以及神经纤维变性，与脑脊液 （CSF） 和血清磷酸化神经丝重链 （pNF-H） 的增加有关。DRGs 的 RNA 测序分析显示，AAV 治疗后，NHPs 和小鼠之间保留了失调的通路，包括先天性/适应性免疫反应的增加以及线粒体和神经元相关基因的减少。最后，内源性 miR-21-5p 在 AAV 处理的 NHP 和小鼠的 DRG 中上调。在 NHP 的 CSF 中还发现了 miR-21-5p 的增加，这与 pNF-H 显著相关，表明 miR-21-5p 与其他分子分析物一起是 DRG 毒性的潜在生物标志物。这项工作强调了在开发用于治疗目的的基于 RNAi 的 AAV 载体时评估与 DRG 毒性相关的安全问题的重要性。